The overall objective of this renewal application is to test the hypothesis that the activation of Trp channel-mediated Ca2+ entry in endothelial cells increases lung microvascular permeability and promotes lung edema. We have shown that thrombin activation of Proteinase-Activated Receptor-1 (PAR-1) expressed on endothelial cell surface induces an increase in intracellular Ca2+, which is critical in the mechanism of increased endothelial permeability. The increase in intracellular Ca2+ concentration ([Ca2+]i) is dependent on both Ca2+ store depletion and the Ca2+ store depletion-mediated Ca2+ influx. We showed that the prevention of Ca2+ influx drastically reduced the thrombin-induced increase in endothelial permeability. We also show that the Ca2+ influx due to store depletion occurs through activation of the transient receptor potential (Trp) gene family of channels expressed in endothelial cells. Our supporting data show that Trp1 (Trp gene homologue) is predominantly expressed in human endothelial cells and increased expression of Trp1 augments the Ca2+ influx in endothelial cells. Further, we show that Src kinase activation plays an important role in the regulation of Ca2+ influx via Trp channels. Deletion of Trp4 gene (predominant Trp isoform expressed in mouse endothelial cells) in mouse impairs thrombin-induced increase in lung microvascular permeability. The association of Trp1 with caveolin-1 (the principal protein of caveolae) in response to thrombin is required for the Ca2+ influx in endothelial cells. We also show that the inflammatory cytokine, tumor necrosis factor-alpha (TNFa) increases Trp1 expression in endothelial cells. Based on these supporting data, we propose to (i) determine the role of Src kinase signaling in the mechanism of Ca2+ influx in endothelial cells via Trp channels (ii) study the role of caveolin-1 in the mechanism of Ca2+ influx through Trp channels and its role in the mechanism of increased endothelial permeability, and (iii) investigate the effects of inflammatory mediators, TNFa and lipopolysaccharide, on the functional expression of Trp channels and its role in the mechanism of increase in endothelial permeability. The proposed studies will utilize cell and in vivo approaches involving Src null (Src -/-), caveolin-1 null (cav1 -/-), and Trp4 null (Trp4 -/-) mice to address the role of Trp channels in activating Ca2+ influx in endothelial cells and in the mechanism of increased endothelial permeability. With the achievement of these studies, we will be able to provide new molecular insight into understanding the mechanism of vascular injury and tissue inflammation associated with states of hypercoagulation such as with sepsis.